The present invention relates to an improved sensor for electronically detecting a binding reaction between molecular structures or a pair of chemical substances, such as oligonucleotides, antigens, enzymes, peptides, antibodies, DNA and RNA fragments.
The present invention further provides a new production method for this improved sensor.
Techniques and sensors for detecting molecular structures and specific substances such as enzywes, peptides, oligonucleotides, antigens antibodies, DNA and RNA fragments in a solution sample are known in the art. In a specific class of sensors, use is made of the principle of measuring the impedance between two electrodes. The absence or presence of DNA-molecules or antibodies or antigens between the electrodes affects the permittivity and/or the conductivity between the electrodes. Various techniques were proposed to measure the presence and/or concentration of a given analyte in a sample solution by using a binding substance element having specific affinity for the analyte. Such specific binding reactions occur e.g. between enzymes and their substrates, antibodies and antigens, between DNA-DNA, between RNA-DNA, or other molecular structures.
Stoner et al in xe2x80x9cAdsorption of blood proteins on metals using capacitance techniquesxe2x80x9d, J. Phys. Chem., 74, Mar. 5, 1970, describe a differential capacity measurement for evaluation of protein adsorption on metalic electrodes.
Arwin et al. in U.S. Pat. No. 4,072,576, use an adsorbed polypeptide substrate and establish a capacitive method for the measurement of enzymatic activity and the immunological interaction assay.
Giaever in U.S. Pat. No. 4,054,646, teaches an electrical method that measures the presence of antibodies in a solution, by coating a metallic substrate with an antigen. After the incubation of the electrodes with the sample solution, he measures capacitively the thickness of the molecular sheet, i.e. he distinguishes between mono- and bimolecular layer, by using a mercury drop as a second electrode.
Newman in Patent application W087/03095 discloses a capacitive sensor for chemical analysis and measurement. Said sensor can be used to detect a broad range of analytes including bacteria, viruses, antibodies, antigens, enzyme substrates and hormones. A thin insulating layer is coated on the surface of conductors and a substrate to form an open capacitor. A biospecific binding agent is immobilized on the surface of the insulating layer between the conductors. The dielectric constant of the biospecific binding agent is altered by binding of the analyte being detected with the biospecific binding agent. A similar sensing principle is disclosed in U.S. Pat. No. 5,114,674.
Battailard et al, 1988, Anal.Chem., 60, 2374-2379 and more recently, Klein et al, 1995, Sensors and Actuators, B 2627, pp. 474-476, show that a metal-semiconductor-insulator device can be used in a similar way as a MIS (metal-insulator-semiconductor) capacitor. The device is immersed in a solution together with a second, reference electrode. By measuring the ac capacity between the metallic layer of the device and the reference electrode, when dc biasing voltages are simultaneously applied, the fiat band voltage of the system is in fact measured in a similar way as in the case of a MIS capacitor. It was proven that the flat band voltage can be modulated by species adsorbed at the insolation-liquid interface. On this principle work the ISFET, ion-sensitive-field-effect-transistor and GENFET, gene-sensitive-field-effect-transistor.
U.S. Pat. No. 4,219,335, issued to Richard Ebersole discusses the use of immune reagents labeled with reactance tags. These tags can be detected ellectrically since they alter the dielectric, conductive or magnetic properties of the test surface.
Simlarly, EP 0 241 771, issued to S. J. Mroczkowski, teaches the detection of metal labeled antibodies by conductometric measurements. When antigens are immobilised inbetween two electrodes, the specific interaction with a metal-labeled antibody is measured by means of resistance decrease of the interelectrode medium.
M. Malmros in U.S. Pat. No. 4,334,880 describes conductivity variation of a semiconductive polymeric layer inbetween two planar electrodes. The said polymer incorporates, in a way or another, certain molecules able to recognize specific analytes. The recognition process induces conductivity changes of the polymeric layer.
Further variations on this central idea of impedimetric sensing appear in the art, EP 0 543 550, EP 0241 771, U.S. Pat. No. 4,453,126, GB 2,137,361, U.S. Pat. No. 3,999,122. Essentially in an impedimetric sensor, certain molecules are immobilised on the top, in between or both on the top and in between a pair of electrodes. Said molecules xe2x80x98recognisexe2x80x99 a specific analyte when exposed to a sample solution This recognition process eventually ends up, directly or indirectly, m conductivity and/or permitivity alteration of the space in the neighbourhood of the electrodes. Finally, by measuring the impedance between the two electrodes, a measure of the recognition process can be established.
The problem associated with the so called sensors as referred to above is that to have a good resolution, the immoblised layer should be perfectly homogenous and should not contain holes, which is hard to achieve.
With the advent of the microelectronic technology, there is a continuous effort to use it in order to develop micro-sensors. Sensors realised with microelectronic technology offer advantages such as low-cost production, increased reproducibility of the production process, uniformity, accurateness of detection, and flexibility in development. Such microelectronic sensors can comprise a multitude of individual test sites with reproducible, uniform electrical properties, whereby enhancing the detection sensitivity of the sensor. The test sites can be made with dimensions of the order of the dimensions of the molecules that have to be detected. The spatial limitations are the fabrication technology resolution and the sensitivity of the device which is dictated by the state of the art in instrumentation and the density of probes. A configuration can also be realised wherein the individual test sites can each yield a different type of signal according to the particular molecule which is to be detected in said test site.
Another important characteristic of the microelectronic technology is its planarity: the microelectrodes patterned this way are essentially flat elements. This feature is not a strong point in the impedimetric devices. In a planar impedimetric structure the electric field lines expand more above the device surface and out of the region of intrest in comparison to real 3-D structures. This is a major drawback especially when the region of interest is limited in space, i.e. it is an enzymatic or polymeric membrane or an adsorbed molecular layer at the surface of the structure. Any field line depassing this region of interest, introduces in the impedimetric response a shunting impedance which can be considered as noise for the measurement.
Still, depending on the electrodes geometry, i.e. dimensions and interspacing, a big majority of the total signal is enclosed in a certain region above the surface of the device as shown in FIG. 1. From the same figure one can deduce that miniaturisation, i.e. L decrease, is crucial in obtaining impedimetric planar structures that probe the space in the very close neighbourhood of the device. An illustration of the dimension down scaling was given by DeSilva et al
DeSilva et al in 1995, Biosensors and Bioelectronics, 10, pp. 675-682 report a new biosensing structure that combines a covalent antibody immobilization technique with a simple impedance response method. The biosensor was fabricated by covalently binding anti-SEB antibodies onto an ultra-thin, island-like, electrically continuous, Pt film deposited onto a silicon chip. They register an impedance decrease when the specific interaction with SEB takes place.
However, the reproducibility is low due to the somewhat random behavior of the fabrication process, i.e. the Pt deposition and the immobilisation procedure.
A true electrode patterning process is likely to insure a good reproducibility of the structures and to improve the control upon sensor behaviour. Devices with patterned features, said features having dimensions of hundreds of nanometers are expected to be highly sensitive to DNA fragments of 300 bases, i.e. Exhibiting a total molecular Tenth of about 180 nm, or to other large molecules like enzymes or antibodies (tens of nanometers diameter). This dimension range is usually achieved in two ways:
1. deep UV or X-ray lithography, techniques where about 100 nm features can be achieved with a fully optimised process.
2. electron-beampatterning, a tedious and very expensive technique where features of tens of nm can be obtained.
It is an aim of the present invention to provide an electrochemical sensor suitable for measuring the presence or absence of molecular structures.
It is another aim of the present invention to provide a method for fabricating as defined above.
It is yet another aim of the present invention to provide a method for detecting the presence of molecular structures in a sample.
The present invention relates to a new electrochemical sensor, based on the interference of an electrical field between electrodes with the analyte. The analyte to be tested is brought in the close neighbourhood of the structure by means of probes.
The present invention relates more particularly to a sensor for identifying molecular structures within a sample solution is disclosed. The sensor comprises an insulating layer with a plurality of interspaced channels therein having essentially the same direction. Said channels have a bottom and at least two opposite side-walls along said direction. The channels furthermore have submicron dimensions. A metal coating is applied on one of said two opposite side-walls of essentially each channel and on top of the insulating layer in between said channels thereby forming an impedimetric device with said sample solution within and between the channels. Optionally probes for binding to said molecular structures are already applied on said sensor. Said probes can be applied to either the insulating part of the channels (said bottom and the other side-wall of said channels), or to the surface of the electrodes or to both, the insulating part of the channels and the surface of the electrodes. Furthermore means are provided for applying a voltage on the metal coatings; as well as means for measuring the impedance in between the electrodes.
The term electrochemical sensor or shortly sensor according to the present invention refers to a device which transforms a (bio)chemical information into an electrical signal.
The present invention overcomes the problem of sensitivity compared to prior art sensors and methods. One important feature of this novel design is the high degree of miniaturisation. This is likely to reduce the noise of the structure and subsequently to increase its sensitivity. Another remarkable feature of the proposed sensor is its tridimensional geometry. This improves the electric field penetration in the area of interest with an eventual sensitivity increase. Said sensor has an interdigitated electrode structure which can be fabricated in a cheap way, even for large active areas.
The probes of the present invention are functionally defined as molecules able to react with another molecule to form a complex an/or induce a secondary reaction. It is by the way of example and not by way of limitation that probes can be enzymes, antibodies, antigens, peptides, DNA fragments, RNA fragments or oligonucleotides. Preferred probes according to the present invention are described in the following patents and patent applications held by one of the present applicants: EP 0 337 896; EP 0 345 375; EP 0 657 532; EP 0 419 355; EP 0 525 095; EP 0 494 317; EP 0489968; EP 0 644 202; WO 92/10514; EP 0 499 003; WO 92/11366; WO 92/16628; WO 92/19770; WO 93/08302; WO 93/18054; EP 0 561 087; WO 93/22437; WO 94/01554; EP 0 637 342; WO 94/13795; WO 94/18325; WO 94/21818; WO 94/25601; WO 95/12666; WO 95/17429; WO 95/33851; WO 96/00298; WO 96/04309; EP 0 721 505; WO 96/13590; WO 96/13608; WO 96/17065; and PCT applications filed under number 96/03091, 96/04146; as well as EP applications filed under Nos. 95870136.9, 96870006.2, 96870081.5, 96870053.4, 96870122.8 or 96870131.8. The contents of these patent (applications) and any other document referred to in this text are to be considered as incorporated by reference. The probes as well as the methods for making these probes are further discussed in the above-mentioned documents. It should be clear that these probes may be purified from a living source or may be made by any method of synthesis known in the art.
The targets to be detected in the sample or analyte can be any molecule present in a sample which binds or reacts with said probes. The targets can thus also include enzymes, antibodies, antigens, peptides, DNA fragments, RNA fragments, oligonucleotides or even whole cells. Depending on the type of targets and type of application, a specific type of recognition circuitry for processing the information related to target detection may be provided with or separately from the sensor.
The sample can be any biological sample (tissue or fluid) containing target molecules to be detected taken directly or after culturing (enrichment) from a healthy or an infected human being or animal More specifically these samples can include expectorations of any kind, blood, plasma, respiratory tract samples such as sputum, broncheolavages, skin tissue, biopsies, lymphoyte blood culture material, colonies, cerebrospinal fluid, brain tissue, urine, gastrointestinal tract, food, feed or environmental samples. Said samples may be prepared or ecxtracted by any method known in the art.
The sample may also be any preparation as described below (such as urea) or any other industrial product.
Alternatively, the sample to be tested may contain partially or fully purified target or analyte molecules, such as for instance amplified nucleotides, which have been solubilized in a solution. These solutions can be chosen from any type of solution known in the art which is suited for establishing a binding reaction between the specific probe and its target.
In the case of nucleotide detection, the sample material will include either genomic DNA or precursor RNA or amplified versions thereof The solution will be what is referred to a as hybridization solution. Upon hybridisation under what is referred to as xe2x80x9cdesired hybridisation characteristics according to the present inventionxe2x80x9d, the probe (in this case an oligonucloetide) will only hybridize to the DNA or RNA from the specific organisms or molecules for which it was designed and not to the DNA or RNA from other organics or molecules such as closely related organisms or variant or mutated molecules which may also be present in a particular sample. In practice, this often implies that the intensity of the hybridization signal is at least two, three, four, five or even ten times stronger with the target DNA or RNA from the organisms from which the probes were designed, as compared to non-target sequences. Often it is desirable and achievable to detect nucleotide which perfectly match the probe nucleotide (implying that hybridization conditions are used in which one mismatch is detectable).
The hybridization conditions can be monitored relying upon several parameters, such as the nature and concentration of the components of the media or solutions, and the temperatures under which the hybrids are formed and washed. When modifications are introduced, be it either in the probes or the media, the temperatures at which the nucleotide probes can be used to obtain the required specificity should be changed according to known relationships, such as those described in Names and Higgins (eds.). Nucleic acid hybridization. A practical approach, IRL Press, Oxford, UK, 1985.
The probes may be applied to the sensor of the present invention in any manner known in the art, for instance immobilized by means of high resolution probe dispensing systems or even synthesized on the spot.
In another set-up also comprised within the scope of the present invention, the sample may be applied to the sensor and the probes may be added in solution to the ative test site area of the sensor to bring about a recognition which may be detected.
It should be stressed that the ability to simultaneously generate recognition results with a number of probes is an outstanding benefit of the sensors of the present invention.
In case of detection of antibodies present in a sample, the probe will be an antigen (e.g. a peptide or a polypeptide) or an anti-idiotype antibody known in the art. In case of detection antigens or polypeptides or peptides possibly present in a sample, the probe will be an antibody or a derivative thereof specifically binding to certain antigens, an antisense peptide specifically binding to certain peptides or polypeptides, a receptor or chemical molecule specifically binding to said polypeptide or peptide. The solution in which the possibly prepared or purified target material present in the sample may be dissolved, will be any solution which allows the binding between said binding molecules to occur. The conditions under which this formation may occur are well known in the art and are for instance further described in the above-mentioned patents and applications of the one of the applicants.
The present invention also relates to method of fabricating a sensor for identifying molecular structures within a sample substance. This method comprises the steps of forming a plurality of interspaced channels in a insulting layer, said channels having essentially the same direction, said channels having a bottom and at least two opposite side-walls along said direction; depositing a metal layer on said insulating layer while aligning said dielectric layer with respect to the metal deposition source such that the bottom of said channels and the side-walls of said canals along the deposition direction are shadowed and not covered by metal to thereby form an impedimetric device with said sample substance within and between the channels and eventually immobilising probes for binding to said molecular structures, said probes being applied to either the insulating part of the channels (said bottom and the other side-wall of said channels), or to the surface of the electrodes or to both, the insulating part of the channels and the surface of the electrodes.
The present invention represents an important tool in a wide field of applications and it is by the way of example and not byway of limitation suited for measuring specific interactions like the reaction between an enzyme and its substrate or the recognition reaction between an antibody and an antigen, between DNA-DNA, between RNA-DNA, or other molecular structures; in the study of the reaction kinetics of said specific interactions; for sequencing molecules such as peptides, enzymes, nucleotides, DNA, RNA and so on; for detecting genes mutations; for epidemiology and geno- or sero typing or for instance HLA and HCV; for drug susceptibility testing like the resistance against beta-lactamase and tetracycline in Neisseria gonorrhoeae, the detection of rifampicin resistant Mycobacterium tuberculosis strains or the detection of AZT-resistance in HIV; in screening and diagnosis~viral diagnosis: like in the case of HIV, HCV, HBV, herpes and relatives, CMV, BPV or HTLV; bacterial diagnosis like in the case of sexually transmitted diseases, cerebral spinal fluid analysis, detection of different mycobacterial species, evaluation of anaerobic infections, otitis, respiratory tract, gastro-intestinal tract, periodontal pathogens, pathogenic fingi; genetic diseases, like cystic fibrosis, Alzheimer, detection of mitochondrial mutations, platelet antigens, drug receptors, risk factors for atherioscrerosis and coronary heart dieases, cancer, APOE, AChE, APOB, LDL and so on; in clinical analysis: like in the case of conductometric urea or creatinine quantitation.
The high sensor miniaturisation also allows the construction of integrated microdiagnostic devices capable of simultaneous detection of a multitude of parameters, i.e. multiparameter testing, and ultimately screening assays.